Identification of the Gram Positive Bacterial Sepsis Agent with Rapid Genotype Test

Year 2021, Volume: 13 Issue: 3, 511 - 518, 18.10.2021

Ziya Erdoğan , Cihadiye Öztürk , Şengül Cangür , Emel Çalışkan , Şükrü Öksüz , Zeynep Karamurat

https://doi.org/10.18521/ktd.834769

Abstract

Objective: An irreversible process begins when a systemic infection causes sepsis. Therefore, rapid identification of the agent bacteria in sepsis and its antibiotic resistance is crucially important. In this study, it was aimed to investigate the efficiency of rapid genotype test.
Methods: Blood culture bottles sent to the laboratory were examined with an automatic blood culture system (BACTEC, BD, USA). Growing bacteria was identificated by VİTEK (bioMérieux, France) automated bacteria identification / antibiotic susceptibility system. In addition, bacterial species and mecA, vanA, vanB, vanC1, vanC2 / C3 genes in blood cultures with Gram positive bacterial growth were also determined by the "Genotype® BC Gram-positive (Hain Lifesience, Germany)" test.
Results: 72 patients with gram-positive bacteria growth in two or more blood culture bottles were included in the study. In 44 of the samples (61%) the same bacterial species were detected with conventional method (bacteria culture) and BC Gram positive test. In 28 of the samples (39%) differences were detected between results of methods regarding bacterial species name or vancomycin/methicillin resistance rate. Although single agent was isolated with culture method in all of the samples, multiple agents were detected in eight samples with rapid genotype test. Also, it was found that in mecA positive samples, ciprofloxacin resistance was higher than mecA negative ones.
Conclusion: In the study, it was observed that BC Gram positive test could correctly identify sepsis agent bacteria and their resistance genes within 4-5 hours.

Keywords

Drug resistance, PCR, Rapid test, Sepsis, Staphylococci

Supporting Institution

DÜBAP

Project Number

017.04.01.618.

Gram Pozitif Bakteriyel Sepsis Etkenlerinin Hızlı Genotip Test ile Tanımlanması

Abstract

Amaç: Sistemik bir enfeksiyon sepsise neden olduğunda geri dönüşü olmayan bir süreç başlamaktadır. Bu nedenle sepsisteki etken bakterinin ve antibiyotik direncinin hızlı bir şekilde tanımlanması çok önemlidir. Bu çalışmada, sepsis etkeni Gram pozitif bakterileri ve önemli antibiyotik dirençlerini saptamada hızlı genotip testinin etkinliğinin araştırılması amaçlanmıştır.
Gereç ve Yöntem: Laboratuvara 2018-2019 yılları arasında gönderilen 2132 kan kültürü örneği otomatik kan kültürü sistemi (BACTEC, BD, ABD) ile incelenmiştir. Üreyen bakteriler VİTEK (bioMérieux,Fransa) otomatize bakteri identifikasyon/antibiyotik duyarlılık sistemi ile tanımlanmıştır. Ayrıca Gram pozitif bakteri üremesi olan kan kültürlerindeki bakteri türleri ve mecA, vanA, vanB, vanC1, vanC2 / C3 genleri “Genotype® BC Gram-pozitif (Hain Lifesience, Almanya)” testi ile de belirlenmiştir.
Bulgular: İki veya daha fazla kan kültürü şişesinde Gram pozitif bakteri üremesi saptanan 72 hasta çalışmaya dahil edildi. Örneklerin 44'ünde (% 61) konvansiyonel yöntemle (bakteri kültürü) ve BC Gram pozitif testi ile aynı bakteri türü tespit edildi. Örneklerin 28'inde (% 39) bakteri tür adı veya vankomisin / metisilin direnç oranı ile ilgili yöntemlerin sonuçları arasında farklılıklar tespit edildi. Tüm örneklerde kültür yöntemi ile tek etken izole edilmesine rağmen hızlı genotip testi ile sekiz örnekte birden fazla etken tespit edildi. Ayrıca mecA pozitif örneklerde siprofloksasin direncinin mecA negatiflere göre daha yüksek olduğu bulundu.
Sonuç: Çalışmada BC Gram pozitif testinin, sepsis etkeni bakterileri ve direnç genlerini 4-5 saat içinde doğru bir şekilde belirleyebildiği görülmüştür.

Keywords

İlaç direnci, PCR, Hızlı test, Sepsis, Stafilokok

Project Number

017.04.01.618.

References

• 1. Gülhan B, Atmaca S, Özekinci T, Suay A. Evaulation of rapid genotype assay fort he identification of Gram-positive cocci from blood cultures and dedection of mecA and van genes. Mikrobiyol Bul. 2011;45(4):592-601.
• 2. Kempf VA, Tiberius K, Autenrieth IB. Fluorescent in sutu hybridization allows rapid identification of microorganism in blood cultures. J Clin Microbiol. 2000;38(2):830-8.
• 3. Eigner U, Weizenegger M, Fahr AM, Witte W. Evaluation of a rapid direct assay for identification of bacteria and the mecA and van genes from positive-testing blood cultures. J Clin Microbiol. 2005;43(10):5256-62.
• 4. Digiovine B, Chenoweth C, Watts C, Higgins M. The attributable mortality and costs of primary nosocomial bloodstream infections in the intensive care unit. Am J Respir Crit Care Med. 1999;160(3):976-81.
• 5. Altun O, Botero-Kleiven S, Carlsson S, Ullberg M, Özenci V. Rapid identification of bacteria from positive blood culture bottles by MALDI-TOF MS following short-term incubation on solid media. J Med Microbiol. 2015;64(11):1346-52.
• 6. She R C, Bender J M. Advances in rapid molecular blood culture diagnostics: Healthcare impact, laboratory implications, and multiplex technologies. The Journal of Applied Laboratory Medicine. 2019;3(4):617-30.
• 7. European Committee on Antimicrobial Susceptibility Testing (EUCAST): 2018. European Committee on Antimicrobial Susceptibility Testing breakpoint tables for interpretation of MICs and zone diameters, version 8.1. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_8.1_Breakpoint_ Tables. pdf.
• 8. WHO Seventieth World Health Assembly, 26 May 2017.
• 9. Minasyan H. Resuscitation and emergency medicine. Scand J Trauma Resusc Emerg Med. 2019;27(19):3-22.
• 10. Global Sepsis Alliance. Misdiagnosed sepsis now a global health priority for World Health Organization, 26 May 2017.
• 11. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34(6):1589-96.
• 12. Cicek A, Kuzucu C, Durmaz B. Factors for interpreting blood culture results. İnönü Üniversitesi Tıp Fakültesi Derg. 2005;12(4):277-80.
• 13. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev. 2010;23(1):235-51.
• 14. Mussap M, Molinari MP, Senno E, Gritti P, Soro B, Mannelli S, et al. New diagnostic tools for neonatal sepsis: The role of a real-time polymerase chain reaction for the early detection and identification of bacterial and fungal species in blood samples. J Chemother. 2007;19(2):31-4.
• 15. Lehmann LE, Hunfeld KP, Emrich T, Haberhausen G, Wissing H, Hoeft A, et al. A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Med Microbiol Immunol. 2008;197(3):313-24.
• 16. Başustaoğlu A(Ed.). Kan kültürü uygulama kılavuzu. Ankara. 2013.
• 17. Bauer M, Reinhart K. Molecular diagnostics of sepsis: Where are we today? Int J Med Microbiol. 2010;300(6):411-3.
• 18. Wolcott MJ. Advances in nucleic acid-based detection methods. Clin Microbiol Rev. 1992;5(4):370-86.
• 19. Prère MF, Baron O, Fayet O. Rapid identification of bacteria, mecA and van genes from blood cultures. Pathol Biol. 2007;55(8-9):375-7.
• 20. Sancak B. Staphylococcus aureus and antibiotic resistance. Mikrobiyol Bul. 2011;45(3):565-76.
• 21. Ippolito G, Leone S, Lauria FN, Nicastri E, Wenzel RP. Methicillin-resistant Staphylococcus aureus: The superbug. Int J Infect Dis 2010;14(4):7-11.
• 22. Stryjewski ME, Corey GR. New treatments for methicillin-resistant Staphylococcus aureus. Curr Opin Crit Care. 2009;15(5):403-12.
• 23. Soriano A, Marco F, Martinez JA, Pisos E, Almela M, Dimova VP, et al. Influence of vancomycin minimum inhibitory concentration on the treatment of methicillin-resistant Staphylococcus aureus bacteremia. Clin Infect Dis. 2008;46(2):193-200.
• 24. Deurenberg RH, Vink C, Kalenic S, Bruggeman CA, Stobberingh EE. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect. 2007;13(3):222-35.
• 25. Stefani S, Goglio A. Methicillin-resistant Staphylococcus aureus: Related infections and antibiotic resistance. Int J Infect Dis. 2010;14(4):19-22.
• 26. Tsiodras S, Gold HS, Sakoulas G, Eliopoulos GM, Wennersten C, Venkataraman L, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet. 2001;358(9277):207-8.
• 27. Steindor M, Weizenegger M, Harrison N, Hirschl AM, Schweickert B, Göbel UB, et al. Use of a commercial PCR-based line blot method for identification of bacterial pathogens and the mecA and van genes from BacTAlert blood culture bottles. J Clin Microbiol. 2012;50(1):157-9.
• 28. Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M, Simor AE. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol. 2002;40(8):2786-90.
• 29. Wilke A, Sayan M, Meriç M, Mutlu B. Early detection of methicillin resistance by Real-Time PCR in staphylococci isolated from blood cultures. Mikrobiyol Bul. 2012;46(4):671-5.
• 30. Sayan M, Meriç M, Celebi S, Willke A. Elimination of PCR inhibitors in routine diagnostic real-time PCR assay and results of internal amplification control. Mikrobiyol Bul. 2009;43(1):179-81.
• 31. Al-Soud WA, Radström P. Purification and characterization of PCR inhibitory components in blood cells. J Clin Microbiol. 2001;39(2):485-93.